Abstract Defining the specific factors that govern the evolution and transmission of influenza A virus (IAV) populations remains a critical open question in virology. IAV populations primarily consist of semi-infectious particles (SIPs) that express highly heterogeneous incomplete subsets of viral genes, and thus depend upon complementation through cellular co-infection to replicate. Viral genomic heterogeneity and co-infection dynamics likely play enormous roles in determining the outcome of IAV infection, yet remain ill-defined. In the proposed studies, we will utilize an array of novel single-virion and single-cell methods to define how genomic heterogeneity influences both co-infection dynamics and the host response to infection. In our preliminary data, we reveal that IAV superinfection, one of the primary mechanisms of viral co-infection, is regulated by the number of viral genes expressed by an infecting virion, rather than the specific activity of the viral neuraminidase gene as has been previously reported. As a consequence, superinfection is more frequent in viral populations that contain more SIPs. In aim 1, we will dissect the specific molecular details of this unique viral gene dose-dependent mechanism of superinfection regulation, as well as determine whether host intrinsic immune mechanisms are involved in regulating superinfection. In aim 2, we will identify the specific viral genetic determinants that regulate superinfection, and test whether IAV strains and mutants that differ in SIP production also vary in superinfection potential as we predict. Finally, our aim 3 studies will build upon our preliminary data that identifies a distinct cluster of host genes that is specifically regulated by the viral neuraminidase gene. We will use single cell RNAseq to leverage the enormous amount of genomic heterogeneity present within IAV populations to dissect the contributions of individual IAV gene segments in determining the overall host transcriptional response to infection. Collectively, these studies will greatly expand our understanding of how genomic heterogeneity within IAV populations shapes the replicative, evolutionary, and pathogenic potential of IAV populations.